Immobilization Of Bacillus Thuringiensis And Applicability In Removal Sulfamethazine From The Soil

Sulfonamides are widely used in animal and poultry industries as typical veterinary drugs because of their stable properties and broad antibacterial spectrum.Most of the sulfonamides ingested by the animal will enter the soil with the animal feces,and then produce toxic effects on the soil microbial community,and can induce the proliferation and transmission of resistance genes.At present,the traditional microbial technology for remediation of sulfonamide antibiotics contaminated soil has the problems of low bacterial colonization rate and poor degradation effect.In view of this situation,this study focused on sulfamethazine(SM2),which is widely used in sulfonamides(SAs),as the research object,using biochar and sodium alginate as the carrier,SM2 degrading bacteria H38 was made into immobilized bacteria.The effects of environmental conditions on SM2 removal performance of immobilized bacteria H38 were studied;the removal effect of immobilized bacteria H38 on SM2 in soil was analyzed;and the functional diversity of soil microbial community,pollution induced community resistance(PICT),and carbon and nitrogen cycle functional genes were analyzed.The integrated biomarker response version2(IBR)was used to evaluate the safety of immobilized H38 in SM2contaminated soil remediation.The main results are as follows:(1)Screening and immobilization of degrading bacteria H38.(1)Screening and identification of degrading bacteria:a total of 6 strains of bacteria with high SM2 degrading ability were screened out from the antibiotic resistant bacteria stored in the laboratory,and the degradation rate of SM2 by 5 strains of bacteria reached 100%on the third day.In the follow-up experiments,H38 with high degradation rate and rapid propagation rate was selected as the experimental bacteria.Combined with 16S rRNA sequence analysis,h38 was identified as Bacillus Thuringiensis.(2)Determination of immobilization conditions:based on pre-experiment,biochar and sodium alginate were selected as the immobilization carrier,and the degradation bacteria were made into immobilized bacteria H38 by the combined technology of adsorption and embedding.The results of degradation rate showed that the immobilized bacteria H38 greatly shortened the time required to degrade SM2 and improved the degradation rate.(2)The characteristics of SM2 removal by immobilized bacteria H38.The results showed that the immobilized strain H38 had a certain impact resistance to a wide range of pH and temperature.When the pH was 5-9 and the temperature was 20-40?,the removal rate of SM2could be maintained above 70%.With the increase of inoculation amount and the decrease of initial concentration of SM2,the removal rate of SM2 increased gradually.Under suitable environmental conditions,SM2 in inorganic salt medium(MSM)can be completely removed by immobilized bacteria H38 within 60 h,while the degradation rate of free bacteria is only75.2%.(3)Application of immobilized bacteria H38 in SM2 contaminated soil.Indoor soil remediation experiments showed that immobilized bacteria h38 could remove 90.0%of SM2in the 12th day,which was higher than that of free bacteria 23.9%in the same period.In addition,the resistance of microbial community to SM2 was significantly reduced by immobilized H38.Compared with the contaminated soil and free bacteria treatment groups,the EC₅₀ of immobilized H38 treatment group decreased by 14.3%and 9.5%respectively.(4)Effects of immobilized bacteria H38 on functional microorganisms in SM2contaminated soil.The results showed that:(1)immobilized bacteria H38 could effectively improve the overall activity of microorganisms in SM2 contaminated soil;compared with the free bacteria treatment group,the Shannon and Simpson index of immobilized bacteria H38treatment group increased significantly,but the Mc Intosh index decreased significantly.(2)The analysis of nitrogen cycling genes showed that compared with contaminated soil and free bacteria treatment,the expression of AOA and AOB was significantly increased in the immobilized bacteria H38 treatment group;similarly,compared with SM2 treatment group and free bacteria treatment group,the expression of cbbLG and cbbM in the immobilized bacteria H38 treatment group was significantly increased,but for cbbLR,the expression of cbbLG in the immobilized bacteria H38 treatment group was significantly lower than that in the free bacteria treatment group Only SM2 treatment group,but significantly higher than free bacteria treatment group.(3)The comprehensive toxicity index analysis showed that the IBR of immobilized bacteria H38 treatment group was the lowest among all SM2 treatment groups.Compared with the contaminated soil and free bacteria treatment groups,the IBR in the immobilized bacteria H38 treatment group decreased by 44.6% and 33.0%,respectively.This shows that immobilized bacteria H38 can reduce the impact of SM2 on soil ecology to a greater extent than free bacteria,and it is a safe and effective repair method.